The present invention relates to the use of novel sapphyrin compounds as photosensitization agents for the photodynamic inactivation of infectious agents having membranous envelopes. The general photodeactivation method used in this example was developed by the Infectious Disease and Advanced Laser Applications Laboratories of the Baylor Research Foundation, Dallas, Texas and is a subject of a U.S. Pat. No. 4,878,891 filed June 25, 1987 by Millard Monroe Judy, James Lester Matthews, Joseph Thomas Newman and Franklin Sogandares-Bernal (assigned to the Baylor Research Foundation, Dallas, Tex.).
For the sake of clarity and brevity, many of the terms used herein have been abbreviated and are set out in the following table:
TABLE 1 ______________________________________ NAME ABBREVIATION ______________________________________ Hepatitis virus Type B HBV Non-A, Non-B hepatitis virus NANB Human lymphototrophic virus type 1 HLTV-1 Human immunodeficiency virus type 1 HIV-1 Simian immunodeficiency virus SIV Herpes simplex virus type 1 HSV-1 dihematoporphyrin ether DHE (commercial form) Photofrin II .RTM. 2,3-diphosphoglycerate 2,3-DPG Vesicular stomatitis virus VSV Phosphate buffered saline PBS Human serum albumin HSA Sodium dodecylsulphate SDS Plaque forming unit per mL PFU/mL L-alpha-phosphatidylcholine PCC Cholesterol C Cholesterol to L-alpha-phosphatidyl- .sup.c /PCC choline ratio Hematoporphyrin derivative HPD Mesochlorin (chlorin of mesoporphyrin) MC Chlorin e6 CE Micromolar uM 3,8,12,13,17,22-hexaethyl-2,7,18,23- Sapphyrin 1 tetramethylsapphyrin 8,17-bis(carboxymethyl)-8,12,13,22- Sapphyrin 2 tetraethyl-2,7,18,23-tetramethyl sapphyrin Adenosine triphosphate ATP Vesicular stomatitis virus VSV ______________________________________
A reliable method of sterilizing or inactivating infectious agents in blood and its products could significantly decrease the risk of transfusion-related disease. The requirement that integrity and full function of blood elements be maintained after prophylaxis imposes severe restrictions upon sterilization methods. To be viable, any blood purification procedure must operate without introducing undesirable toxins, damaging normal blood components, or inducing the formation of harmful metabolites. In general, this precludes the use of common antiviral systems such as those based on heating, UV irradiation, or purely chemical means. A photodynamic inactivation approach is a promising approach obviating many of these disadvantages.
Alternatively, blood could be collected into dye-coated bags, and irradiated during the normal post-collection sedimentation period. The bags might be plastic or some other suitable material to which the sapphyrin compound would bind without losing its ability to react with molecular oxygen upon irradiation.
In connection with photodynamic inactivation, a porphyrin compound, dihematoporphyrin ether, was studied by Schnipper et al. (Schnipper, L. E., Lewin, A. A., Swartz, M., and Crumpacker, C. S. (1980) "Mechanisms of photodynamic inactivation of herpes simplex viruses," J. Clin. Invest., 65, 432-438), and shown to act as an efficient photosensitizer for the photoinactivation of both cell-free and cell infected enveloped viruses (Skiles, H. M., Judy, M., Newman, J. T. (1987) "photodynamic inactivation of viruses with hematoporphyrin derivatives" Abstr. of 6th Southern Biomedical Engineering Conference, 1987, 83). It is likely that the success of this procedure derives from the fact that this dye localizes selectively at or near the morphologically characteristic and physiologically essential viral membrane "envelope" (which has no direct counterpart in normal blood elements) and catalyzes the formation of singlet oxygen upon photoirradiation. The singlet oxygen so produced is believed, in turn, to destroy the essential membrane envelope, thus killing the virus and eliminating its infectivity. Photodynamic blood purification procedures, therefore, apparently rely on the use of photosensitizers which localize selectively at viral membranes.
However, "first generation" dyes such as DHE are not ideal and suffer from a number of serious deficiencies. They contain a range of chemical species, are neither catabolized nor excreted rapidly from the body, and absorb poorly in the red part of the spectrum where blood and body tissues are most transparent (van Gemert, M. J. C., Welch, A. J., Amin, A. P. (1986) "Is there an optimal laser treatment for port wine stains?" Lasers Surg. Med. 6, 76-83). Each of these deficiencies can and does have important clinical consequences.
Effective concentrations can and often do vary from preparation to preparation because of the fact that DHE and its analogs do not contain a single chemically well-defined constituent and the active components have yet to be identified with certainty. Significant quantities of these dyes may remain in patients after treatment because they are not rapidly metabolized. In fact DHE is known to localize in the skin and to induce severe and extended photosensitivity in patients (Oseroff, A. R., Ohuoha, D., Ara, G., McAuliffe, D., Foley, J., Cincotta, L. Proc. Natl. Acad. Sci. USA, (1986), 83, 9729 and references therein). Finally because the longest wavelength absorption maximum for these dyes falls at 630 nm, most of the incipient energy used is dispersed or attenuated before reaching a blood-borne pathogen. As a result, less of the initial light is available for singlet oxygen production and photodynamic action (van Gemert, M. J. C., Welch, A. J., Amin, A. P. (1986) "Is there an optimal laser treatment for port wine stains?" Lasers Surg. Med. 6, 76-83). Thus the development of photosensitizers absorbing in the 700 nm region is desirable, provided desirable features such as selective localization on pathogens, low dark toxicity, and efficient photosensitization, are maintained.
The photodynamic inactivation of infectious agents using visible light range photosensitizers is emerging as a potential means of sterilizing banked blood and its products (Skiles, H., Judy, M. M., Newman, J. T. (1985) "Photodynamic inactivation of viruses with hematoporphyrin derivatives", Abstr. Am. Soc. for Microbiol., p. 7, A 38; Skiles, H., Judy, M. M., Newman, J. T. (1987) "Photodynamic inactivation of viruses with hematoporphyrin derivatives", Abstr. of 6th Southern Biomedical Engineering Conference, 1987, 83; Matthews, J. L., Newman, J. T., Sogandares-Bernal, F., Judy, M. M., Skiles, H. Leveson, J. E., Marengo-Rowe, A. J., Chanh, T. C. (1988) "Photodynamic therapy of viral contaminants with potential for blood banking applications", Transfusion, 28, 81-83 (Rapid Communication); Matthews, J. L., Sogandares-Bernal, F., Judy, M. M., Marengo-Rowe, A. J., Skiles, H., Leveson, J., Chanh, T., and Newman, J. (1988) "Photodynamic inactivation of human immunodeficiency virus in human blood", Transfusion, 28(S), 31S; Dennis, M. V., Judy, M. M., Matthews, J. L. and Sogandares-Bernal, F. (1989) "Protective qualities of mitochondrial and cytosolic fluorescent dyes against in vitro and in vivo infection by the tulahuen strain of Trypanosoma cruzi, J. Parasitol. (Accepted for Publication); Chanh, T., Allan, J., Matthews, J. L., Sogandares-Bernal, F., Judy, M. M., Newman, J. T. (1989) "Photodynamic inactivation of simian immunodeficiency virus in blood", (Accepted for publication by Exp. Virol); Sieber, F. (1988) "Antineoplastic and antriviral properties of merocyanine 540", in SPIE, Advances in Photochemotherapy (Edited by H. Tayaba) 997, 128). The enveloped viruses, HIV and its simian analogue (SIV), and HSV-1, all suspended in whole human blood have been inactivated using DHE (Matthews, J. L., Newman, J. T., Sogandares-Bernal, F., Judy, M. M., Skiles, H. Leveson, J. E., Marengo-Rowe, A. J., Chanh, T. C. (1988), "Photodynamic therapy of viral contaminants with potential for blood banking applications", Transfusion, 28, 81-83 (Rapid Communication); Chanh, T., Allan, J., Matthews, J. L., Sogandares-Bernal, F., Judy, M. M., Newman, J. T. (1989), "Photodynamic inactivation of simian immunodeficiency virus in blood", (Accepted for publication by Exp. Virol). No measurable changes in red blood cell membrane integrity, 2,3-DPG activity concentration, or in ATP concentration followed photodynamic treatment of whole blood containing 200 ug/ml of DHE (estimated 333 uM macrocycle concentration) (Matthews, J. L., Sogandares-Bernal, F., Judy, M. M., Marengo-Rowe, A. J., Skiles, H., Leveson, J., Chanh, T., and Newman, J. (1988) "Photodynamic inactivation of human immunodeficiency virus in human blood", Transfusion, 28(S), 31S). This concentration of DHE is estimated to exceed (by a factor of 10-20) that required for viral inactivation.
In 1985, an estimated 10 million units of whole blood were processed by over 800 blood banks in the U.S. and 14 million units of blood components were transfused. This use reflects major needs for blood components in managing trauma, hemorrhagic and neoplastic disorders, and recipients of bone marrow or solid organ transplants. Use of blood products still involves significant risk to the recipient because of the potential transmission of infectious agents. Among human infections, viruses which are enclosed by a lipid membrane or envelope, such as the hepatitis viruses (HBV and NANB), HLTV-1 (a leukemia virus), and HIV-1 (the AIDS virus), as well as Chagas' disease and malaria can be transmitted by blood transfusion (Cohen, M. D., Munoz, A., Reitz, B. A., et al. (1989) "Transmission of retroviruses by transfusion of screened blood in patients undergoing cardiac surgery", N. Engl. J. Med. 320, 1172-1176). Screening of donors and serologic testing reduce the risk, but these precautions still provide insufficient protection as detectable HIV-1 antibody may not be present during the early stage of infection (Cohen, M. D., Munoz, A., Reitz, B. A., et al. (1989) "Transmission of retroviruses by transfusion of screened blood in patients undergoing cardiac surgery", N. Engl. J. Med. 320, 1172-1176).
In a number of prior studies on photodynamic inactivation of viruses and protozoan infectious agents in blood (Skiles, H., Judy, M. M., Newman, J. T. (1985) "Photodynamic inactivation of viruses with hematoporphyrin derivatives", Abstr. Am. Soc. for Microbiol., p. 7, A 38; Skiles, H., Judy, M. M., Newman, J. T. (1987) "Photodynamic inactivation of viruses with hematoporphyrin derivatives", Abstr. of 6th Southern Biomedical Engineering Conference, 1987, 83; Matthews, J. L., Newman, J. T., Sogandares-Bernal, F., Judy, M. M., Skiles, H. Leveson, J. E., Marengo-Rowe, A. J., Chanh, T. C. (1988) "Photodynamic therapy of viral contaminants with potential for blood banking applications", Transfusion , 28, 81-83 (Rapid Communication); (Matthews, J. L., Sogandares-Bernal, F., Judy, M. M., Marengo-Rowe, A. J., Skiles, H., Leveson, J., Chanh, T., and Newman, J. (1988) "Photodynamic inactivation of human immunodeficiency virus in human blood", Transfusion, 28(S), 31S; Dennis, M. V., Judy, M. M., Matthews, J. L. and Sogandares-Bernal, F. (1989) "Protective qualities of mitochondrial and cytosolic fluorescent dyes against in vitro and in vivo infection by the tulahuen strain of Trypanosoma cruzi, J. Parasitol. (Accepted for Publication); Chanh, T., Allan, J., Matthews, J. L., Sogandares-Bernal, F., Judy, M. M., Newman, J. T. (1989) "Photodynamic inactivation of simian immunodeficiency virus in blood." (Accepted for publication by Exp. Virol). Photofrin II.RTM. and excitation with 630 nm light were used. Blood appreciably absorbs as well as scatters light in the wavelength region &lt;630 nm (e.g. see FIG. 1 of van Gemert et al. 1986), and it exhibits a broad relative absorption minimum at approximately 680 nm. Therefore, efficient photosensitizers absorbing in the longer red wavelength region potentially offer more efficient use of excitation light energy. Photophysical measurements of Maiya, B. G., Cyr, M., Harriman, A., and Sessler, J. L. (1989) "In-vitro photodynamic activity of diprotonated sapphyrin: a 22 pi-electron pentapyrrolic porphyrin-like macrocycle", (Submitted to J. Phys. Chem.) have shown that the triplet state of decaalkylsaphyrin macrocycle (FIG. 1, Structure 1) in monomer form efficiently generates singlet oxygen (Phi.sub.delta =0.25 CH.sub.3 OH). As the sapphyrins also exhibit extinction coefficients in the range of 1.times.10.sup.4 cm.sup.-1 at wavelengths near 680 nm (See FIG. 2) and offer potential as long wavelength photosensitizers, the photodynamic inactivation of cell-free, enveloped HSV-1 using the free-base sapphyrin (FIG. 1, Structure 1) and its dicarboxyl functionalized analogue (FIG. 1, Structure 2) was studied. In order to assess qualitatively their degree of monomerization and to identify the viral binding environment of the photosensitizer, fluorescence measurements were made of these sapphyrins dissolved in solvents of different polarity or in the presence of liposomes, human plasma proteins, and cell-free VSV (also an enveloped virus).
Consequently, the present inventors have discovered that sapphyrins are effective photosensitizers for the photo-eradication of cell-free viruses, and especially cell-free HIV-1. The sapphyrin compounds are new and no sapphyrins have been used for this purpose before. One of the sapphyrins tested is the single most effective substance yet found for the photodynamic eradication of the AIDS virus, being twice as efficient on a normalized per macrocycle incident unit of light basis as the current best available dye, DHE.